Unlimited Cooperativity of Betatectivirus SSB, a Novel DNA Binding Protein Related to an Atypical Group of SSBs From Protein-Primed Replicating Bacterial Viruses.

Ana Lechuga,Darius Kazlauskas,Modesto Redrejo-Rodríguez,Margarita Salas

doi:10.3389/fmicb.2021.699140

Abstract

Bam35 and related betatectiviruses are tail-less bacteriophages that prey on members of the Bacillus cereus group. These temperate viruses replicate their linear genome by a protein-primed mechanism. In this work, we have identified and characterized the product of the viral ORF2 as a single-stranded DNA binding protein (hereafter B35SSB). B35SSB binds ssDNA with great preference over dsDNA or RNA in a sequence-independent, highly cooperative manner that results in a non-specific stimulation of DNA replication. We have also identified several aromatic and basic residues, involved in base-stacking and electrostatic interactions, respectively, that are required for effective protein–ssDNA interaction. Although SSBs are essential for DNA replication in all domains of life as well as many viruses, they are very diverse proteins. However, most SSBs share a common structural domain, named OB-fold. Protein-primed viruses could constitute an exception, as no OB-fold DNA binding protein has been reported. Based on databases searches as well as phylogenetic and structural analyses, we showed that B35SSB belongs to a novel and independent group of SSBs. This group contains proteins encoded by protein-primed viral genomes from unrelated viruses, spanning betatectiviruses and Φ29 and close podoviruses, and they share a conserved pattern of secondary structure. Sensitive searches and structural predictions indicate that B35SSB contains a conserved domain resembling a divergent OB-fold, which would constitute the first occurrence of an OB-fold-like domain in a protein-primed genome.

Highlights

The Bacillus virus Bam35 is the model virus of the genus Betatectivirus, a group of temperate Tectiviridae members infecting Gram-positive animal and human pathogens from the Bacillus cereus group (Ackermann et al, 1978; Gillis et al, 2018)
The Escherichia coli SSB (EcoSSB) monomer has a similar molecular weight to B35SSB (19 and 18.5 kDa, respectively), EcoSSB-single-stranded DNA (ssDNA) complexes showed higher mobility than B35SSB-ssDNA complexes, which indicates that a higher number of B35SSB molecules are bound to the ssDNA fragment
We found that B35SSB has a high specificity for ssDNA over dsDNA and RNA

Summary

Introduction

The Bacillus virus Bam is the model virus of the genus Betatectivirus, a group of temperate Tectiviridae members infecting Gram-positive animal and human pathogens from the Bacillus cereus group (Ackermann et al, 1978; Gillis et al, 2018). The genome of Bam is replicated by a protein-priming DNA replication process (Berjón-Otero et al, 2016), a widespread mechanism for the initiation of genome replication in a number of linear genomes of viruses and linear plasmids. By this mechanism, a specific amino acid of the so-called terminal protein (TP) primes the replication providing a hydroxyl group for the incorporation of the first nucleotide by the viral DNA polymerase and it becomes covalently linked to the 5 genome ends. Not identified in Bam, a number of accessory DNA binding proteins, such as singlestranded DNA binding proteins (SSBs), increase the efficiency of genome replication in vitro and are essential in vivo in the case of 29 and other systems (Pakula et al, 1990; Salas, 1991; Blanco et al, 1994)

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